The techniques of combinatorial chemistry have been increasingly exploited in the process of drug discovery. Combinatorial chemistry allows for the synthesis of a wide range of compounds with varied molecular characteristics. Combinatorial synthetic techniques enable the synthesis of hundreds to millions of distinct chemical compounds in the same amount of time required to synthesize one or a few compounds by classical synthetic methods. Subjecting these compounds to high-throughput screening allows thousands of compounds to be rapidly tested for desired activity, again saving time expense and effort in the laboratory.
Chemical combinatorial libraries are diverse collections of molecular compounds. Gordon et al. (1995) Acc. Chem. Res. 29:144-154. These compounds are formed using a multistep synthetic route, wherein a series of different chemical modules can be inserted at any particular step in the route. By performing the synthetic route multiple times in parallel, each possible permutation of the chemical modules can be constructed. The result is the rapid synthesis of hundreds, thousands, or even millions of different structures within a chemical class.
For several reasons, the initial work in combinitorial library construction focused on peptide synthesis. Furka et al. (1991) Int. J. Peptide Protein Res. 37:487-493; Houghton et al. (1985) Proc. Natl. Acad. Sci. USA 82:5131-5135; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81:3998-4002; Fodor et al, (1991) Science 251:767. The rapid synthesis of discrete chemical entities is enhanced where the need to purify synthetic intermediates is minimized or eliminated; synthesis on a solid support serves this function. Construction of peptides on a solid support is well-known and well-documented. Obtaining a large number of structurally diverse molecules through combinatorial synthesis is furthered where many different chemical modules are readily available; hundreds of natural and unnatural amino acid modules are commercially available. Finally, many peptides are biologically active, making them interesting as a class to the pharmaceutical industry.
The scope of combinatorial chemistry libraries has recently been expanded beyond peptide synthesis. Polycarbamate and N-substituted glycine libraries have been synthesized in an attempt to produce libraries containing chemical entities that are similar to peptides in structure, but possess enhanced proteolytic stability, absorption and pharmacokinetic properties. Cho et al. (1993) Science 261:1303-1305; and Simon et al. (1992) Proc. Natl. Acad. Sci. USA 89, 9367-9371. Furthermore, benzodiazepine, pyrrolidine, and diketopiperazine libraries have been synthesized, expanding combinatorial chemistry to include heterocyclic entities. Bunin et al. (1992) J. Am. Chem. Soc. 114:10997-10998; Murpy et al. (1995) J. Am. Chem. Soc. 117:7029-7030; and Gordon et al. (1995) Biorg. Medicinal Chem. Lett. 5:47-50.
Hydroxylamines and their derivatives, including hydroxamic acids, hydroxyl ureas, and hydroxyl sulfonamides, have been the subject of much research focused on their properties as metalloproteinase inhibitors. Izquierdo-Martin et al. (1992) J. Am. Chem. Soc. 114:325-331; and Cushman et al. (1981) Chapter 5 "Specific Inhibitors of Zinc Metallopeptidases" in Topics in Molecular Pharmacology (Burgen & Roberts, eds.).
Metalloproteinases are members of a superfamily of enzymes which share a number of features. Their activity depends on the peptide nature of their substrates; full enzymatic activity requires a metal ion (generally zinc, cobalt, or iron) bound by the side chains of conserved amino acids at or near the active site; among the conserved metal-binding residues are histidines belonging to a motif, HEXXH. The enzyems are sensitive to metal-chelating reagents. Vallee and Auld (1990) Biochem. 29:5647-5659; and Stocker et al. (1995) Protein Sci. 4: 823-840. Currently, the family comprises two subclasses, exemplified by thermolysin and metzincins.
The metalloproteinase superfamily encompasses metalloproteinases from a wide variety of organisms. For example, matrix metalloproteinases in mammals act to modify or degrade extracellular matrix components such as collagens, fibronectin, and laminin. Birkedal-Hansen et al. (1993) Crit. Rev. Oral Biol. Med. 4:197-250. MMP's are believed to be involved in the development of arthritis, tumor angiogenesis, retinopathy, and many other disease processes. While many MMP's are secreted from the cell, others remain membrane bound. Takino et al. (1995) J. Biol. Chem. 270:23013-23020; Will and Hinzmann (1995) Eur. J. Biochem. 231:602-608; and Tuner and Tanzawa (1997) FASEB J. 11:355-364. Other metalloproteinases isolated from mammals include endopeptidase EC 3.4.24.15, which is believed to be involved in the regulated metabolism of a number of neuropeptides (Papastoitsis et al. (1994) Biochem. 33:192-199; and McDermott et al. (1992) Biochem. Biophys. Res. Comm. 185:746-753); angiotensin-converting enzyme; endothelin-converting enzyme; and neutral endopeptidase. Homologues of these various human metalloproteinases have been reported in a variety of animal species. Snake venom metalloproteinases also degrade major proteins of the extracellular matrix, and further have been reported to degrade platelet integrin VLA-2 and von Willebrand factor. Jia et al. (1996) Toxicon 34:1269-1276; and Kamiguti et al. (1996) Toxicon 34:627-642. Fungi such as Aspergillus and Fusarium have been reported to synthesize metalloproteinases. Sekine (1973) Agric. Biol. Chem. 37:1945-1952; and U.S. Pat. No. 5,691,162. Metalloproteinases have also been isolated from parasitic organisms which can be pathogenic toward mammals, including protozoan parasites such as helminths (U.S. Pat. No. 5,691,186). Bacteria also synthesize metalloproteinases. Hase et al. (1993) Microbiol. Rev. 57:823-837. Metalloproteinases have been isolated from various bacteria including Bacillus species such as Bacillus subtilis (McConn et al. (1964) J. Biol. Chem. 239:3706); Serratia (Miyata et al. (1971) Agr. Biol. Chem. 35:460); Legionella pneumophila (Moffat et al. (1994) Infection and Immunity 62:751-753); Vibrio species (Takahashi et al. (1996) Biosci. Biotech. Biochem. 60:1651-1654; and Clostridium species such as Clostridium perfringens (Minami et al. (1997) Microbiol. Immunol. 41:527-535. Activities of some of these enzymes can produce deleterious effects in mammals. For example, the .lambda.-toxin of C. perfringens acts to cleave and activate another toxin produced by this bacterium. Minami et al. (1997). Other bacterial metalloproteinases can act to activate zymogen forms of human MMP's. Okamoto et al. (1997) J. Biol. Chem. 272:6059-6066.
A relatively new member of the metalloproteinase superfamily is the bacterial enzyme peptide deformylase (PDF). In bacteria, nascent proteins typically contain an N-formyl group on the N-terminal methionine. This enzyme catalyzes removal of the formyl moiety from nascent proteins, and this activity is essential for maturation of nascent proteins. Deformylase activity is critical to the growth of Escherichia coli. Chang et al. (1989) J. Bacteriol. 171:4071-4072; and Meinnel and Blanquet (1994) J. Bacteriol. 176:7387-7390. While this enzyme clearly shares many of the features which characterize metalloproteinases, it differs from other members of the superfamily in several important respects. Firstly, the metal ion in the active enzyme appears to be Fe(II), or possibly another divalent cationic metal, instead of the zinc ion more commonly encountered. Rajagopalan et al., (1997) J. Am. Chem. Soc., 119:12418-19. Secondly, the divalent ion appears to play an important role, not only in catalysis, but also in the structural integrity of the protein; thirdly, the third ligand of the divalent ion is a cysteine, rather than a histidine or a glutamate, as in other metalloproteinases; fourthly, this third ligand is not located at the C-terminal side of the HEXXH motif but far away along the amino acid sequence, and N-terminal to the motif; finally, the solution structure shows significant differences in the secondary and tertiary structure of PDF, compared with other prototypical metalloproteinases. Meinnel et al. (1996) J. Mol. Biol. 262:375-386. PDF from E. coli, Bacillus stearothermophilus, and Thermus thermophilus have been characterized. Meinnel et al. (1997) J. Mol. Biol. 267:749-761. The enzyme studied by Meinnel et al. contained a zinc ion as the divalent ion, and the structural features summarized above were obtained from zinc-containing proteins.
U.S. Pat. No. 5,268,384 discloses hydroxamates and hydroxyl ureas used to treat angiogenesis by inhibiting matrix metalloproteinases. Among metalloproteinases disclosed as targets of inhibitors are collagenases, including human skin fibroblast collagenase and purulent human sputum collagenase; gelatinases, including human skin fibroblast gelatinase and purulent human sputum gelatinase; and stromelysin. Disclosed disorders amenable to treatment by matrix metalloproteinase (MMP) inhibitors include ocular pathologies such as diabetic retinopathy and neovascular glaucoma; cancer, including Kaposi's sarcoma, glioblastoma, and angiosarcoma; immune system disorders such as rheumatoid arthritis; and skin disorders such as psoriasis.
Patent publication WO 96/26918 discloses hydroxamates for inhibiting MMPs. The publication also discusses the inhibition of the production or the action of the cytokine tumor necrosis factor (TNF) by hydroxamic acid MMP inhibitors. See also, Mohler et al. Nature 370:218-220 (1994); Gearing et al., Nature 370:555-557 (1994); and McGeehan et al., Nature 370:558-561 (1994). These MMP inhibitors are described as useful for treating inflammatory, infectious, immunological or malignant diseases due to their effect on TNF. Among the specific diseases described are septic shock, hemodynamic shock, malaria, meningitis, fibrotic disease, cachexia, autoimmune diseases, and graft rejection.
Patent publication WO 96/25156 discloses hydroxamates for inhibiting matrix metalloproteinases. The publication also discusses inhibition of production or processing of transforming growth factor alpha (TGF-.alpha.) by MMP inhibitors, and describes potential applications of the MMP inhibitors in treating inflammation; wound healing, including scar and keloid formation; diabetic retinopathy; neovascular glaucoma; atherosclerosis; vascular adhesions; systemic lupus erythrematosus; various carcinomas; and other diseases amenable to treatment by modulating production or processing of TGF-.alpha..
U.S. Pat. No. 5,552,419 discloses aryl sulfonamido-substituted hydroxamic acids. The compounds are described as inhibitors of stromelysin, gelatinase and/or collagenase. Disorders described as amenable to treatment by the hydroxamic acid derivatives are osteoarthritis and rheumatoid arthritis; tissue ulceration; periodontal disease; bone diseases, including Paget's disease and osteoporosis; HIV infection; and tumor metastasis, tumor progression or tumor invasion.
Patent publication EP 423943 describes the use of inhibitors of certain matrix metalloproteinases, such as collagenases, gelatinases, and stromelysins, as useful for treatment of demyelinating diseases such as multiple sclerosis and other scleroses; demyelinating peripheral neuropathies; acute disseminated encephalomyelitis; and other neural disorders.
Other hydroxamic acid-based metalloproteinase inhibitors are described in the following patent publications: U.S. Pat. Nos. 4,599,361 and 5,256,657; European patent publications EP 236872, EP 274453, EP 489577, EP 489579, EP 497192, EP 574758; and international PCT applications WO 90/05716, WO 90/05719, WO 91/02716, WO 92/13831, WO 92/22523, WO 93/09090, WO 93/09097, WO 93/20047, WO 93/24449, WO 93/24475, WO 94/02446, WO 94/02447, WO 94/21612, WO 94/25434, and WO 94/25435.
Many synthetic routes to produce hydroxylamines have been developed and are well-known in the art (see the above-cited publications for representative examples). These methods are limited by the necessity of preparing one compound at a time. Solid-phase synthesis of an immobilized hydroxamate is mentioned in patent application WO 96/26918; however, the method used in the application is limited to the Ugi reaction described. See also, Strocker et al. Tet. Lett. 37:1149-1152 (1996); Keating et al., J. Am. Chem. Soc. 118:2574-2583 (1996); and Tempest et al. Angew. Chem. Int. Ed. Engl. 35:640-642 (1995), and references therein.
The invention disclosed herein provides a method for combinatorial synthesis of hydroxylamines and hydroxylamine derivatives, enabling synthesis of a much greater variety of compounds in a relatively short amount of time.
All references, publications and patents mentioned herein are hereby incorporated herein in their entirety.